Regeneration of Pinus halepensis (Mill.) through Organogenesis from Apical Shoot Buds

Organogenesis and somatic embryogenesis have been widely applied as the two main regeneration pathways in plant tissue cultures. However, recalcitrance is still the main restriction in the clonal propagation of many woody species, especially in conifers. They undergo a "phase change" that leads to significant loss of vegetative propagation capacity, reducing the aptitude of tissues and organs to be regenerated in vitro beyond this point. In line with this, the in vitro regeneration of mature conifer trees has been a long-cherished goal in many laboratories worldwide. Based on previous works in Pinus species regeneration from adult trees, we now present data about the culture of apical shoot buds in an attempt to induce organogenesis and somatic embryogenesis to clone mature trees of Aleppo pine (Pinus halepensis). Reinvigorated axillary shoots were submitted to conditions usually applied to induce somatic embryogenesis through the manipulation of culture media, including the use of auxins such as 2,4-Dichlorophenoxyacetic acid and 1-Naphthaleneacetic acid, cytokinins (6-benzyladenine and kinetin), and phytosulfokine (50, 100, and 200 nM). Although somatic embryos could not be obtained, an embryogenic-like tissue was produced, followed by the emergence of actively proliferating non-embryogenic calli. Variations in the consistence, texture, and color of non-embryogenic calli were observed; especially those arising in the media containing phytosulfokine. Reinvigorated shoots, induced by 22 or 44 mu M 6-benzyladenine, were obtained through organogenesis and acclimatized, and phenotypically normal plants were obtained.